This application is submitted in response to PA-09-199 Identifying non-coding RNA (ncRNA) targets for cancer early detection and prevention (R01). Our objective is to develop sputum-based small ncRNA biomarkers that can combine with the genomic probes to improve early detection of lung squamous cell cancer (LSqCC). LSqCC is the second most frequent lung cancer. In 2010, LSqCC will cause an estimated 47,190 deaths, accounting for 8.4 % of all cancer deaths in the USA. Given the poor prognosis associated with advanced stage LSqCC, its early detection will potentially reduce the mortality. However, the current diagnostic techniques for LSqCC early detection are either invasive or have poor accuracy. We have collected induced sputum samples from early stage LSqCC patients and cancer-free controls. We developed an enrichment technique that can efficiently collect deep respiratory epithelial cells from the sputum samples. We also developed sputum-based genomic probes that can diagnose LSqCC with higher sensitivity compared with sputum cytology. However, the sensitivity of the probes is not high enough for clinical application. Small ncRNAs are emerging as potential biomarkers in cancer diagnosis. We have been the first to demonstrate that miRNAs are stably present in sputum. Furthermore, using microarrays to analyze 42 early stage LSqCC tissues and paired noncancerous lung tissues, we recently identified 39 LSqCC-associated small ncRNAs. The ncRNAs include 30 miRNAs and 9 snoRNAs, providing new potential biomarkers for diagnosis of the malignancy. Therefore, we hypothesize that combining the ncRNA biomarkers with the genomic probes in sputum will improve early detection of LSqCC. Taking advantage of our well-characterized induced sputum samples of the cases and controls, we will perform the following four specific aims: 1), determining whether the miRNAs and snoRNAs can be accurately and robustly measured in sputum by qRT-PCR, 2), optimizing a panel of highly specific and sensitive ncRNA biomarkers for early stage LSqCC in a training set of the sputum specimens, 3), validating the ncRNA biomarkers in an testing set of the samples, and 4), evaluating the combined use of the ncRNA biomarkers and genomic probes for early detection of LSqCC. Future application of the biomarkers in clinical settings will potentially provide a useful means to improve early detection of LSqCC, and hence reduce the mortality. The study will strongly support one of the PA's main interests Combination of ncRNA and genetic variation markers in body fluids to increase accuracy of cancer diagnosis.